Received August 24, 2017; Revised October 17, 2017; Accepted October 29, 2017.

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Based on these findings, we collected 84 cacti samples from cactus farms and fields in four provinces (Gyeonggi, Gangwon, Chungcheong and Jeju) in South Korea and investigated the co-infection of cactus plants with various viruses by electron microscopy and RT-PCR analysis from previously unpublished studies. In the investigated cactus plants, only Notocactus leninghausii f. cristatus, obtained from a cactus farm in Gyeonggi province, was co-infected with various viruses. In this study, we identified the viral co-infections in N. leninghausii f. cristatus. The N. leninghausii f. cristatus did not exhibit the typical virus symptoms of mosaic and mottle, chlorotic spots, necrosis, stunting, or distortion. Electron microscopy analysis was performed using a method modified from that described by Min et al. (2006) at NISEM (Seoul National University, http://www.nicem.snu.ac.kr/). Here, crude cactus sap was used for analysis rather than purified virus as described in the original method. In the sap of asymptomatic N. leninghausii f. cristatus (Fig. 1A), we were able to observe virus particles, such as rod-shaped and filamentous virions (Fig. 1B) typical of tobamovirus (320 nm in length and 18 nm in width) and potexvirus (580 nm in length and 13 nm in width), respectively, as previously reported (Maliareko and Mudrak, 2013; Milbrath et al., 1973; Min et al., 2006).

To confirm co-infection with tobamovirus and potexvirus in N. leninghausii f. cristatus, specific primers for detecting the gene encoding the coat proteins (CPs) of two tobamoviruses (CMMoV and RCNaV) and five potexviruses (CVX, OpVX, PiVX, SchVX and ZyVX) were designed using the NCBI nucleotide database (NC_011803, NC_002815, LC128411, JF937699, NC_006060, NC_024458, NC_016442, KU854932, NC_011659, KU854929, KP090203, NC_006059, JF930326) with Lasergene software (DNA Star Inc., Wl, USA) (Table 1). Total RNA of the cactus plants was used for RT-PCR analysis. Total RNA was purified using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, followed by a reverse transcription (RT) reaction using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Pittsburgh, PA, USA). PCR products were amplified using a thermocycler (MyCycler, Bio-Rad, Hercules, CA, USA) with the following program: initial denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 30 s, annealing at 55–58°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 10 min. The annealing temperatures used were determined according to Table 1.

Following RT-PCR analysis, two tobamoviruses (CMMoV and RCNaV) and four potexviruses (CVX, PiVX, SchVX, and ZyVX) were detected in the N. leninghausii f. cristatus sample; OpVX was not detected (Fig. 1C). RT-PCR products for detection of CMMoV and RCNaV were observed at the expected target sizes of 486 and 513 bp, respectively, as were those for detection of CVX (678 bp), PiVX (678 bp), SchVX (678 bp), and ZyVX (681 bp) (Table 1, Fig. 1C). Additionally, roughly spherical cactus-infecting viruses, such as TSWV and SCV, were not observed via electron microscopy analysis and were not detected via RT-PCR using specific TSWV CP and SCV CP primers (data not shown).

To confirm the identification of the detected viruses in the N. leninghausii f. cristatus, DNA products obtained by RT-PCR were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) and sequenced by Bioneer Inc (Daejeon, Korea). Sequences were compared with previously reported nucleotide sequences in the NCBI BLAST database. Nucleotide and amino acid sequences were analyzed using DNAMAN software version 5.1 (Lynnon Biosoft, San Ramon, CA, USA). The viral sequences obtained in this study were given the strain name Nl derived from N. leninghausii and were classified into tobamoviruses and potexviruses.

Most cactus-infecting viruses, such as CVX, OpVX, SOV, SCV, and TSWV, have been reported in the USA, which is one of the countries that exports cacti into South Korea. CMMoV, CVX, and RCNaV were previously reported in Gymnocalycium mihanovichii, Hylocereus spp., and Aporcactus flagelliformis, respectively, in South Korea (Kim et al., 2012; Min et al., 2006). Grafting is generally used to reproduce and propagate cactus plants, but viruses can be transmitted easily through this method (Bausher, 2013; Estrada-Luna et al., 2002; de Medeiros et al., 2006; Maliareko and Mudrak, 2013; Min et al., 2006). The N. leninghausii f. cristatus collected from the cactus farms in this study was also produced using the grafting method. Consequently, co-infection with various viruses (six species) in N. leninghausii f. cristatus may be related to grafting. This study demonstrates that N. leninghausii f. cristatus can be co-infected with at least six different viruses (CMMoV, CVX, RCNaV, PiVX, SchVX, and ZyVX).

Acknowledgments

This study was supported by a sabbatical year research grant from Seoul Women’s University (2016), and by grants (2014M3A9B8022821M3A9B8022015R1D1A1A01060614) from the National Research Foundation in Korea.